Volume 4, Issue 3, September – October 2010; Article 015
ISSN 0976 – 044X
DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF
RISPERIDONE AND TRIHEXYPHENIDYL HYDROCHLORIDE IN TABLET DOSAGE FORMS
Dhara Patel*1, Jayvadan Patel2
Research Scholar of Singhania University, Pacheri bari, Jhunjhunu, Rajasthan, Gujarat, India.
2
Nootan Pharmacy College, S.P. Sahkar Vidhyadham, Kamana Crossing, Visnagar - 384315, Mehsana, Gujarat, India.
*Email: patel.dhara.j@gmail.com
1
ABSTRACT
A binary mixture of Risperidone (RIS) and Trihexyphenidyl hydrochloride (THP) was determined using reversed-phase liquid
chromatography method using methanol: acetonitrile: 0.05 M phosphate buffer (pH 3.7) (60:30:10, v/v/v) pumped at a flow rate of 1.0
ml/min. Quantification was achieved with ultraviolet detection at 230 nm over concentration ranges of 0.5-5.0 and 1.0-10.0 µg/ml;
mean accuracies were 100.55±0.64 and 100.75±0.81%, respectively. The method was successively applied to tablet dosage forms as no
chromatographic interferences from the tablet excipients were observed. The method retained its accuracy and precision when the
standard addition technique was applied.
Keywords: Risperidone, Trihexyphenidyl hydrochloride, RP-HPLC.
INTRODUCTION
Risperidone (RIS) is a psychotropic agent belonging to the
chemical class of benzisoxazole derivatives. Chemically it
is 3-[2-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)-1-piperidinyl]
ethyl]-6, 7, 8, 9-tetrahydro- 2-methyl-4H-pyrido [1, 2-a]
pyrimidin-4-one1-2. It is indicated for the acute and
maintenance treatment of schizophrenia in adolescents
aged 13-17 years and also it is indicated for the shortterm treatment of acute manic or mixed episodes
associated with Bipolar Disorder in adults and in children
and adolescents aged 10-17 years3-4. Trihexyphenidyl
(THP) is an antidyskinetic and antiparkinson drug whose
IUPAC name is 1-cyclohexyl-1-phenyl-3-(1-piperidyl)-1propanol1. THP is official in IP. IP suggest a titrimetric
assay method for THP. Literature survey revealed that
HPLC, UV and HPTLC methods5-28 have been reported for
the estimation of RIS and THP individually and with other
drugs in pharmaceutical dosage forms. RIS and THP are
formulated together in the form of a tablet. Literature
survey revealed no method reported for simultaneous
determination of the two drugs. The present RP-HPLC
method uses simple mobile phase ratio, higher sensitivity
and analysis will complete before 6 min. Therefore the
present study was to determine both drugs concurrently
by sensitive, accurate, rapid and precise RP-HPLC method
for routine analysis.
MATERIALS AND METHODS
The chromatography was performed on a Shimadzu
(Columbia, MD) RP-HPLC instrument (LC-2010 CHT)
equipped with PDA detector, Phenomenex (Torrance, CA)
C18 column (250×4.6 mm id, 5 µm particle size) was used
as stationary phase. Standard samples of RIS and THP and
Market samples of Sizdon forten tablets (Sun Pharma),
each tablet contained 4mg RIS and 2 mg THP were used.
Triple distilled water, methanol, acetonitrile (HPLC grade,
S. D. Fine Chemical, Ahmedabad, India), dihydrogen
phosphate (AR grade, S. D. Fine Chemical, Ahmedabad,
India) were used.
RIS and THP stock solutions (50 µg/ml and 100 µg/ml,
respectively) were prepared by weighing accurately 2.5
mg RIS and 5.0 mg THP powder into 2 separate 50 ml
volumetric flasks; 25 ml methanol was added, shaken for
a few minutes, and diluted to volume with methanol.
From these solutions (2.5 ml) were transferred into 2
separate 10 ml volumetric flasks and diluted to the mark
with methanol to give final concentrations of 12.5 and
25.0 µg/ml, respectively. Accurate aliquots equivalent to
0.5-5.0 µg RIS from its working solution (12.5 µg/ml) and
aliquots equivalent to 1.0-10.0 µg THP from its working
solution (25 µg/ml) were transferred into 2 separate sets
of 5 ml volumetric flasks and diluted to volume with
methanol. Powder from the mixed contents of 20 tablets,
equivalent to 4 mg RIS and 2 mg THP, was transferred
accurately to a 50 ml volumetric flask and diluted to
volume with methanol. The solution was diluted to the
same concentrations of working standard solutions and
treated according to the linearity for the RP-HPLC
method. The separation was done on a C18 column using
methanol: acetonitrile: 0.05 M phosphate buffer (pH 3.7)
(60:30:10, v/v/v) the mobile phase pumped at a flow rate
of 1.0 ml/min. The chromatogram was recorded under
the following instrumental parameters: 10 µl injection
volume, flow rate, 1.0 ml/min at 400C temperature and
the eluent monitored at 230 nm. Calibration curves for
both RIS and THP were plotted, and the corresponding
regressions Eqns were calculated.
RESULTS AND DISCUSSION
The aim of this work was to develop sensitive, accurate,
precise and rapid analytical method for the simultaneous
determination of RIS and THP. This was achieved using
RP-HPLC method. To optimize the proposed RP-HPLC
method, all of the experimental conditions were
International Journal of Pharmaceutical Sciences Review and Research
Available online at www.globalresearchonline.net
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Volume 4, Issue 3, September – October 2010; Article 015
ISSN 0976 – 044X
investigated. For the choice of the stationary phase,
reversed-phase separation was preferred due to the
drawbacks of the normal phase, e.g., hydration of silica
with water that can cause peak tailing. To optimize the
mobile phase, different systems were tried for
chromatographic separation of the two components by
combining homogenous design and solvent polarity
optimization. The best resolution was achieved using a
mobile phase consisting of methanol: acetonitrile: 0.05 M
phosphate buffer (pH 3.7) (60:30:10, v/v/v), which gave
good resolution and sensitivity of both drugs (fig. 1).
Table 1: System suitability test parameters for RIS & THP
for proposed RP-HPLC method
Proposed methods
RP-HPLC
a
a
RIS± % RSD
THP± % RSD
3.797±0.11
5.389± 0.18
Parameters
Retention time, min
Tailing factor
Asymmetry factor
Theoretical plates
Repeatability of
b
measurement (n = 6)
1.15±0.16
1.09±0.51
4253.8±0.64
1.31±0.52
1.20±0.18
6293.98±1.34
1.82
1.49
a
RSD is a Relative standard deviation,
determinations
Figure 1: RP-HPLC chromatogram of RIS and THP at 230 nm
System suitability testing of the RP-HPLC method gave
good relative retention time = 1.592; theoretical plates =
4253.8 and 6293.98; asymmetry factor (A) = 1.09 and
1.20; and tailing factor (T) = 1.15 and 1.31 for RIS and
THP, respectively (Table 1).
b
n is number of
A linear relation was obtained between peak area and the
concentration of the two drugs in the range of 0.5-5.0 and
1.0-10.0 µg/ml for RIS and THP, respectively. The linear
regressions Eqns were computed as: Y=154926 X + 11501,
r= 0.9995 and Y=126789 X+ 45913, r= 0.9979, where Y is
the area under the peak, X is the concentration in µg/ml,
and r is the correlation coefficient. Results obtained by
applying the RP-HPLC procedure showed that RIS and THP
can be simultaneously analyzed in the prepared mixtures
with mean recoveries of 100.55±0.64 and 100.75±0.81 %,
respectively. The proposed method has been applied to
assay RIS and THP in tablets without any interference
from the additives (Table 2). The limit of detection for RIS
and THP were found to be 0.05µg/ml and 0.5µg/ml,
respectively; the limit of quantification for RIS and THP
were found to be 0.5µg/ml and 1.0 µg/ml, respectively by
visual method. The low % CV values of intra-day (0.151.41 for RIS and 0.46-1.38 for THP) and inter-day (0.191.67 for RIS and 0.37-1.62 for THP) precision reveal that
the proposed method is precise. Thus, the proposed
procedure can be used in routine analysis.
Table 2: Assay results for tablets using the proposed methods (RP-HPLC)
Mix.
a
Amount of drug added (mg)
Amount of drug found (mg)
% Amount found (na=3) ± SDb
RIS
THP
RIS
THP
RIS
THP
1
4
2
4.02
1.99
100.50.19
99.50.16
2
4
2
3.97
2.01
99.250.44
100.50.21
n is number of determinations, SD is a Standard deviation, RIS is Risperidone, THP is Trihexyphenidyl HCl
b
Table 3: Application of the standard addition technique to the analysis of RIS and THP in tablets by the proposed methods
Proposed
methods
RP-HPLC
Amount of
drug taken
(µg/ml or ng/spot)
Amount of
drug added
(µg/ml or ng/spot)
Amount of drug
found (µg/ml or
ng/spot)
a
b
% Recovery (n =3) ± SD
RIS
THP
RIS
THP
RIS
THP
RIS
THP
1
2
0.5
2
1.504
4.04
100.31±0.50
100.72±0.79
1
2
1
4
2.014
6.01
100.7±1.16
100.79±0.58
2
1.5
6
2.516
8.51
100.66±0.22
b
n is number of determinations, SD is a Standard deviation
100.63±1.07
1
a
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Volume 4, Issue 3, September – October 2010; Article 015
CONCLUSION
We have successfully developed a new simple RP-HPLC
method for the simultaneous estimation of Risperidone
and Trihexyphenidyl HCl combination in mixture using
simple mobile phase buffer, methanol and acetonitrile.
Rapidity and capability of qualifying very low
concentration of respective drugs, made them useful for
variety of analyses, including pure drug analysis, assay of
formulations and stability studies analysis. The purposed
method did not utilize any extraction step for recovering
the drug from the formulation excipient matrixes and
their by decreased the degree of error, time in estimation
of the drugs and the overall cost of the analysis. The
method was validated and found to be simple, sensitive,
accurate, precise and economical. The proposed method
could be applied for routine analysis in quality control
laboratories.
Acknowledgement: The authors are grateful to Sun
Pharmaceuticals Ltd (Vadoadara, Gujarat, India) for the
gift samples of pure RIS and THP, respectively. The
authors would also like to thank anonymous reviewers for
their helpful comments. Of course, all remaining errors
are mine.
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